Double-strand DNA breaks (DSBs), which can result from exposure of cells to various environmental DNA damaging agents, including ionizing radiation, ultraviolet light and various chemicals, are a potent source of the genetic instabilities that fuel neurological diseases and cancer in humans. In particular, long-tract repair DNA synthesis (LTRS) that is essential for completion of several DSB repair pathways, including break- induced replication (BIR) and long-tract gene conversion (LTGC), frequently leads to mutations and chromo- somal rearrangements. In yeast, when combined with DNA damage sources, including alkylating agents or APOBEC enzymes, LTRS leads to massive genomic destabilization events that mimic kataegis and chro- mothripsis associated with cancer in humans. Due to the lack of convenient experimental systems, the mo- lecular mechanism of LTRS in mammalian cells remains unexplored. In addition, the identity of mammalian proteins that drive LTRS and those that suppress LTRS remain unknown, which precludes an adequate understanding of how genetic stability is maintained in mammalian cells and how genomic instability leading to cancer can be unleashed. The goal of this proposed research is to characterize the proteins that suppress LTRS in mammalian cells as well as those driving LTRS. To this end, several sensitive mammalian cell reporters have been developed to enable detection of LTRS by reconstitution of either fluorescent protein- encoding genes or by formation of genes encoding antibiotic resistance. LTRS suppressors will be identified using a candidate-gene approach in addition to a whole-genome shRNA screen using reporter cells trans- formed with the shRNA library and analyzed via next-generation sequencing to identify shRNAs that stimulate LTRS events. Identification of LTRS drivers in mammalian cells will be aided by the results of a successful whole-genome screen for LTRS drivers that has been performed in yeast and led to identification of multiple gene candidates, including genes encoding topoisomerase, helicases, chromatin remodeling proteins, tran- scription factors, and mitotic spindle checkpoint factors. The proposed research will characterize the respec- tive roles of these proteins in DSB repair in yeast and will determine the role of their mammalian homologs in LTRS in mammals. Overall, the results of this research project will increase our basic knowledge of DSB repair mechanisms and causes of genetic instability associated with DSB repair in eukaryotes and in mam- mals in particular. In addition, this research will yield sensitive biosensors that can be used for assessing the efficiency of LTRS and its contribution to genetic instability across numerous cell line models and in the context of a variety of environmental perturbagens. In addition, identification of mammalian LTRS suppres- sors and drivers will contribute to an increased understanding of cancer etiology.